Note: Descriptions are shown in the official language in which they were submitted.
~`` ORTH 355
~L1'7~33
HYBRID CELL LINE FO~ PRODUCING MONOCLONAL ANTIBODY TO
HUMAN CYTOTOXIC AND SUPPRESSOR T CELLS, ANTIBODY, AND
MET~ODS
_
FIELD OF THE INVENTION
This invention relates generally to new hybrid cell lines
and more specifically to hybrid cell lines for production
of monoclonal antibody to an antigen found on normal human
cytotoxic and suppressor T cells, to the antibody so
produced, and to therapeutic and diagnostic methods and
compositions employing this antibody.
DESCRIPTION OF THE PRIOR ART
The fusion of mouse myeloma cells to spleen cells from
immunized mice by Rohler and Milstein in 1975 [Nature 256,
495-497 (1975)] demonstrated for the first time that it
was possible to obtain a continuous cell line making
homogeneous (so-called "monoclonal") antibody. Since this
seminal work, much effort has been directed to the
production of various hybrid cells (called "hybridomas")
and to the use of the antibody made by these hybridomas
for various scientific investigations. See, for example,
Current ToPics in Microbiologv and ImmunologY, Volume 81 -
NLymphocyte Hybridomasn, F. Melchers, M. Potter, and N.
Warner, Editors, Springer-Verlag, 1978, and references
contained therein; C. J. Barnstable, et al., Cell, 14,
9-20 (May, 1978); P. Parham and W. F. Bodmer, Nature 276,
397-399 (November, 1978); Handbook of E~rimental
Immunologv, Third Edition, Volume 2, D. M. Wier, Editor,
Blackwell, 1978, Chapter 25; and Chemical and Engineering
News, January 1, 1979, 15-17. These references simultan-
eously indicate the rewards and complications af attempt-
ing to produce monoclonal antibody from hybridomas. While
the general technique is well understood conceptually,
there are many difficulties met and variations required
for each specific case. In fact, there is no assurance,
- - ORTH 355
~7(~55'3
prior to attempting to prepare a given hybridoma, that the
desired hybridoma will be obtained, that it will produce
antibody if obtained, or that the antibody so produced
will have the desired speci~icity. The degree o~ success
is influenced principally by the type of antigen employed
and the selection technique used for isolating the desired
hybridoma.
The attempted production of monoclonal antibody to human
lymphocyte cell-surface antigens has been reported only in
a few instances. See, for examplet Current Topics ln
MicrobiologY and Immunology, ibid, 66-69 and 164-169. The
antigens used in these reported experiments were cultured
human lymphoblastoid leukemia and human chronic
lymphocytic leukemia cell lines. Many hybridomas obtained
appeared to produce antibody to various antigens on all
human cells. None of the hybridomas produced antibody
against a predefined class of human lymphocytes.
It should be understood that there are two principal
classes of lymphocytes involved in the immune system of
humans and animals. The first of these (the thymus-
derived cell or T cell) is differentiated in the thymus
from haemopoietic stem cells. While within the thymus,
the differentiating cells are termed "thymocytes." The
mature T cells emerge from the thymus and circulate
between the tissues, lymphatics, and the bloodstream.
These T cells form a large proportion of the pool of
recirculating small lymphocytes. They have immunological
specificity and are directly involved in cell-mediated
immune responses (such as graft rejection) as effector
cells. Although T cells do not secrete humoral
antibodies, they are sometimes required for the secretion
of these antibodies by the second class of lymphocytes
discussed below. Some types of T cells play a regulating
function in other aspects of the immune system.
~ ORT~ 355
117C~
The mechanism of this process of cell cooperation is not
yet completely understood.
The second class of lymphocytes (the bone marrow-derived
cells or B cells) are those which secrete antibody. They
also develop from haemopoietic stem cells, but their
differentiation is not determined by the thymus. In
birds, they are differentiated in an organ analogous to
the thymus, called the Bursa of Fabricius. In mammals,
however, no equivalent organ has been discovered, and it
is thought that these B cells differentiate within the
bone marrow.
It is now recognized that T cells are divided into at
least several subtypes, termed "helper", "suppressor", and
~killer" T cells, which have the function of (respectively)
promoting a reaction, suppressing a reaction, or killinq
(lysing) foreign cells. These subclasses are well
understood for murine systems, but they have only recently
been descri~ed for human systems. See, for example, R.
L. Evans, et al., Journàl of Experimental Medicine, Volume
145, 221-232, 1977; and L. Chess and S. F. Schlossman -
~F~nctional Analysis of Distinct Human T-Cell Subsets
Bearing Unique Differentiation Antigens~, in "Contemporarv
ToPics in ImmunobiologYn, O. Stutman, Editor, Plenum
Press, 1977, Volume 7, 363-379.
The ability to identify or suppress classes or subclasses
of T cells is important for diagnosis or treatment of
various immunoregulatory disorders or conditions.
For example, certain leukemias and lymphomas have
differing prognosis depending on whether they are o B
cell or T cell origin. Thus, evaluation of the disease
prognosis depends upon distinguishing between these two
~ 355
:~17(~S~
classes of lymphocytes. See, for example, A. C. Aisenberg
and J. C. Long, The American Journal of Medicine, 58.300
(March, 1975); D. Belpomme, et al., in "Immunologlcal
Diagnosis of Leukemias and Lvmphomasn, S. Thierfelder, et
al., eds, Springer, Heidelberg, 1977, 33-45; and D.
Belpomme, et al., British Journal of Haematology, 1978,
38, 85.
Certain disease states (e.g., juvenile rheumatoid arthri-
tis, malignancies, and agammaglobulinemia) are associated
with an imbalance of T cell subclasses. It has been
suggested that autoimmune diseases generally are associ-
ated with an excess of ~helper" T cells or a deficiency of
certain "suppressor" T cells, while agammaglobulinemia is
associated with an excess of certain "suppressor" T cells
or a deficiency of "helper~ T cells. Malignancies
generally are associated with an excess of "suppressor" T
cells.
In certain leukemias, excess T cells are produced in an
arrested stage of development. Diagnosis may thus depend
on the ability to detect this imbalance or excess. See,
for example, J. Kersey, et al., "Surface Mar~ers Define
Human Lymphoid Malignancies with Differing Prognoses" in
HaematologY and Blood Transfusion, Volume 20, Springer-
Verlag, 1977, 17-24, and references contained therein.
Acquired agammaglobulinemia, a disease state in which no
immune globulin is produced, comprises at least two
distinct types. In type I the failure to produce immune
globulin is due to an excess of suppressor T cells, while
in type II it is due to a lack of helper T cells. In both
types, there appears to be no defect or lack in the
patients' ~ cells, the lymphocytes which are responsible
for the actual secretion of the antibody; however, these B
cells are being either suppressed or "not helped",
~- ORTH 355
1~7~ 3
resulting in greatly decreased or absent immune globulin
production. The type of acquired aqammaglobulinemia may
thus be determined by testing for an excess of suppressor
T cells or an absence of helper T cells.
On the therapeutic side, there is some suggestion, as yet
not definitely proven, that administration of antibodies
against the subtype of T cell in excess may have thera-
peutic benefit in autoimmune disease or malignancies. For
example, a helper T cell cancer (certain cutaneous T cell
lymphomas and certain T cell acute lymphoblastic
leukemias) may be treated by an antibody to a helper T
cell antigen. Treatment of autoimmune disease caused by
an excess of helper cells may also be accomplished in the
same fashion. Treatment of diseases (e.g., malignancies
or type I acquired agammaglobulinemia) due to an excess of
suppressor T cells may be treated by administration of an
antibody to a suppressor T cell antigen.
Antisera against the entire class of human T cells
(so-called antihuman thymocyte globulin or ATG) has been
reported useful therapeutically in patients receiving
organ transplants. Since the cell-mediated immune
response ~the mechanism whereby transplants are rejected)
depends upon T cells, administration of antibody to T
cells prevents or retards this rejection process. See,
for example, Cosimi, et al., "Randomized Clinical Trial of
ATG in Cadaver Renal Allgraft Recipients: Importance of T
Cell ~onitoringn, SurgerY 40:155-163 (1976) and references
contained therein.
The identi~ication and suppression of human T cell classes
and subclasses has previously been accomplished by the use
of spontaneous autoantibodies or selective antisera ~or
human T cells obtained by immunizing animals with human T
cells, bleeding the animals to obtain serum, and adsorbing
the antiserum with (for example) autologous but not
,.
~ ~` ORT~ 355
117G~93
allogeneic B cells to remove antibodies with unwanted
reactivities. The preparation of these antisera is
extremely difficult, particularly in the adsorption and
purification steps. Even the adsorbed and purified
antisera contain many impurities in addition to the
desired antibody, for several reasons. First, the serum
contains millions of antibody molecules even before the T
cell immunization. Second, the immunization causes
production of antibodies against a variety of antigens
found on all human T cells injected. There is no selec-
tive production of antibody against a single antigen.
Third, the titer of speci~ic antibody obtained by such
methods is usually quite low, (e.g., inactive at dilutions
greater than 1:100) and the ratio of specific to
non-specific antibody is less than 1/10
See, for example, the Chess and Schlossman article refer-
red to above ~at pages 365 and following) and the Chémical
and Engineering News article referred to above, where the
deficiencies of prior art antisera and the advantages of
monoclonal antibody are described.
One of the subsets of T cells identiied by such prior art
antisera has been designated the TH2+ subset and has been
shown to contain both the cytotoxic effector cells ~or
cell-mediated lympholysis and the immunoregulatory
suppressor T cells which suppress both T cell and 3 cell
function. This subset contains about 20%-30~ of human
peripheral T cells. See, for example, articles by E. L.
Reinherz, et al., in J. Immunol. 123:83~1979) and New
Engl. J. Med. 300:1061(1979).
SUMMARY OF THE INVENTION
There has now been discovered a novel hybridoma (designa-
ted OKT5) which is capable of producing novel monoclonal
antibody against an antigen found on normal human periph-
eral cytotoxic and suppressor TH2+ T cells (about 203 of
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7~5~3
normal human peripheral T cells). The antibody 50
produced is monospecific for a single determinant on
normal human cytotoxic and suppressor TH2~T cells and
contains essentially no other anti-human immune globulin,
in contrast to prior art antisera (which are inherently
contaminated with antibody reactive to numerous human
antigens) and to prior art monoclonal antibodies (which
are not monospecific for a human cytotoxic/suppressor T
cell antigen). Moreover, this hybridoma can be cultured
to produce antibody without the necessity of immunizing
and killing animals, followed by the tedious adsorption
and purification steps necessary to obtain even the impure
antisera of the prior art.
It is accordingly one object of this invention to provide
hybridomas which produce antibodies against an antigen
found on normal human cytotoxic and suppressor TH2+ T
cells.
It is a further aspect of the present invention to provide
methods for preparlng these hybridomas.
A further object of the invention i9 to provide essential-
ly homogeneous antibody against an antigen found on normal
human cytotoxic and suppressor TH2+ T cells.
A still further object is to provide methods for treatment
or diagnosis of disease employing these antibodies.
Other objects and advantages of the invention will become
apparent from the examination of the present disclosure.
In satisfaction of the foregoing objects and advantages,
there is provided by this invention a novel hybridoma
producing novel antibody to an antigen found on normal
human cytotoxic and suppressor TH2+ T cells, the antibody
itself, and diagnostic and therapeutic methods employing
~ 593 ORT~ 355
the antibody. The hybridoma was prepared generally fol-
lowing the method of Milstein and Kohler. Following
immunization of mice with normal human thymocytes, the
spleen cells of the immunized mice were fused with cells
from a mouse myeloma line and the resultant hybridomas
screened for those with supernatants containing antibody
which gave selective binding to normal E rosette positive
human T cells. The desired hybridomas were subsequently
cloned and characterized. As a result, a hybridoma was
obtained which produces antibody (designated OKT5) against
an antigen on normal human cytotoxic and suppressor TH2+ T
cells. Not only does this antibody react with normal
human peripheral cytotoxic and suppressor TH2+ T cells,
but it also does not react with other normal peripheral
blood lymphoid cells, including helper T cells. In
addition, the cell surface antigen recognized by this
antibody is detected on approximately 80% of normal human
thymocytes.
In view of the difficulties indicated in the prior art and
the lack of success reported using malignant cell lines as
the antigen, it was surprlsing that the present method
provided the desired hybrldoma. It should be emphasized
that the unpredlctable nature of hybrid cell preparation
does not allow one to extrapolate from one antigen or cell
system to another. In fact, the present applicants have
discovered that using a T cell malignant cell line as the
antigen caused formation of hybridomas which did not
produce the desired antibody. Attempts to use purified
antigens separated from the cell surfaces were also
unsuccessful.
~oth the subject hybridoma and the antibody produced
thereby are identified herein by the designation "OKT5",
the particular material referred to being apparent from
the context.
,,
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S~3
The preparation and characterization of the hybridoma and
the resultant antibody will be better understood by refer-
ence to the following description and Examples.
DETAILED DESCRIPTION OF THE INVENTION
The method o~ preparing the hybridoma generally comprises
the ~ollowing steps:
A. Immunizing mice with normal human thymocytes. While
it has been found that female CAFl mice are preferred, it
is contemplated that other mouse strains could be used.
The immunization schedule and thymocyte concentration
should be such as to produce useful quantities of suitably
primed splenocytes. Three immunizations at fourteen day
intervals with 2 x 107 cells/mouse/injection in 0.2 ml
phosphate buffered saline has been found to be effective.
B. Removing the spleens from the immunized mice and
making a spleen suspension in an appropriate medium.
About one ml of medium per spleen is sufficient. These
experimental techniques are well-known.
C. Fusing the suspended spleen cells with mouse myeloma
cells from a suitable cell line by the use of a suitable
fusion promoter. The preferred ratio i5 about 5 spleen
cells per myeloma cell. A total volume of about 0.5 - 1.0
ml of fusion medium is appropriate for about 10
splenocytes. Many mouse myeloma cell lines are known and
available, generally from members of the academic
community or various deposit banks, such as the Salk
Institute Cell Distribution Center, La Jolla, CA. The
cell line used should preferably be of the so-called "drug
resistant" type, so that unfused myeloma cells will not
survive in a selective medium, while hybrids will survive.
The most common class is 8-azaguanine resistant cell
lines, which lack the enzyme hypoxanthine guanine
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~17~iS93
phophoribosyl transferase and hence will not be supported
by HAT (hypoxanthine, aminopterin, and thymidine) medium.
It is also generally preferred that the myeloma cell line
used be of the so-called "non-secreting" type, in that it
does not itself produce any antibody, although secreting
types may be used. In certain cases, however, secreting
myeloma lines may be preferred. While the preferred
fusion promoter is polyethylene glycol having an average
molecular weight from about 1000 to about 4000
(commercially available as PEG 1000, etc.), other fusion
promoters known in the art may be employed.
D. Diluting and culturing in separate containers, the
lS mixture of unfused spleen cells, unfused myeloma cells,
and fused cells in a selective medium which will not
support the unfused myeloma cells for a time sufficient to
allow death of the unfused cells (about one week). The
dilution may be a type of limiting one, in which the
volume of diluent is statistically calculated to isolate a
certain number of cells (e.g., 1-4) in each separate
container (e.g., each well of a microtiter plate). The
medium is one (e.g., HAT medium) which will not support
the drug resistant (e.g., 8-azaguanine resistant) unfused
myeloma cell line. Hence, these myeloma cells perish.
Since the unfused spleen cells are non-malignant, they
have only a finite number of generations. Thus, after a
certain period of time (about one week) these unfused
spleen cells fail to reproduce. The fused cells, on the
other hand, continue to reproduce because they possess the
malignant quality of the myeloma parent and the ability to
survive in the selective medium of the spleen cell
parent.
1~7C 5~3 O~.T~ 355
E. Evaluating the supernatant in each container (well)
containing a hybridoma for the presence of antibody to E
rosette positive purified human T cells.
F. Selecting (e.g., by limiting dilution) and cloning
hybridomas producing the desired antibody.
Once the desired hybridoma has been selected and cloned,
the resultant antibody may be produced in one of two ways.
The purest monoclonal antibody is produced by ln vitro
culturing of the desired hybridoma in a suitable medium
for a suitable length of time, followed by recovery of the
desired antibody from the supernatant. The suitable
medium and suitable length of culturing time are known or
are readily determined. This ln vitro technique produces
essentially monospecific monoclonal antibody, essentially
free from other specific antihuman immune globulin. There
is a small amount of other immune globulin present since
the medium contains xenogeneic serum (e.g., fetal calf
serum). However, this in vitro method may not produce a
sufficient quantity or concentration of antibody for some
purposes, since the concentration of monoclonal antibody
i8 only about 50 ~/ml.
To produce a much greater concentration of slightly less
pure monoclonal antibody, the desired hybridoma may be
in~ected into mice, preferably syngenic or semi-syngenic
mice. The hybridoma will cause formation of antibody-
producing tumors after a suitable incubation time, which
will result in a high concentration of the desired
antibody (about 5-20 mg/ml) in the bloodstream and
peritoneal exudate ~ascites) of the host mouse. Although
these ho~t mice also have normal antibodies in their blood
and ascites, the concentration of these normal antibodies
is only about 5% of the monoclonal antibody concentration.
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~7~S93
12
Moreover, since'these normal antibodies are not antihuman
in their specificity, the monoclonal antibody obtained
from the harvested ascites or from the serum is essential-
ly free of any contaminating antihuman immune globulin.
This monoclonal antibody is high titer (active at dilu-
tions of l:50,000 or higher) and high ratio of specific to
non-specific immune globulin (about l/20). Immune globu-
lin produced incorporating the ~ light myeloma chains are
non-specific, "nonsense~ peptides which merely dilute the
monoclonal antibody without detracting from its specific-
ity.
EXAMPLE I
Production of Monoclonal Antibodies
.
A. Immunization and Somatic Cell HYbridization
Female CAFl mice (JacXson Laboratories; 6-8 weeks old)
were immunized intraperitoneally with 2 x 107 human
thymocytes in 0.2 ml of phosphate buffered saline at
14-day lntervals. Four days after the third immunization,
spleens were removed from the mice, and a single cell
suspension was made by pressing the tissue through a
stainless steel mesh.
Cell fusion was carried out according to the procedure
developed by Kohler and Milstein. 1 x 108 splenocytes
were fused in 0.5 ml of a fusion medium comprising 35%
polyethylene glycol (PEG 1000) and 5% dimethylsulfoxide in
RPMI 1640 medium (Gibco, Grand Island, NY) with 2 x 107
P3X63Ag8Ul myeloma cells supplied by Dr. M. Scharf f,
Albert Einstein College of Medicine, Bronx, NY. These
myeloma cells secrete Ig~ ~ light chains.
B. Selection and Growth of HYbridoma
After cell fusion, cells were cultured in HAT medium
(hypoxanthine, aminopterin, and thymidine) at 37C with ~
C2 in a humid atmosphere. Several weeks later, 40 to
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117(~S~3
13
100~1 of supernatant from cultures containing hybridomas
were added to a pellet o~ I06 peripheral lymphocytes
separated into E rosette positive (E ) and E rosette
negative ~E ) populations, which were prepared from blood
of healthy human donors as described by Mendes (J.
Immunol. 111:860, 1973). Detection of mouse hybridoma
antibodies binding to these cells was determined by
indirect immunofluorescence. Cells incubated with culture
supernatants were stained with a fluorescinated
goat-anti-mouse IgG (G/M FITC) (Meloy Laboratories,
Springfield, VA; F/p = 2.5) and the fluorescent
antibody-coated cells were subsequently analyzed on the
Cytofluorograf FC200/4800A (Ortho Instruments, Westwood,
MA) as described in Example III. Hybridoma cultures
containing antibodies reacting specifically with E
lymphocytes (T cells) were selected and cloned twice by
limiting dilution methods in the presence of feeder cells.
Subsequently, the clones were transfe r red
intraperitoneally by injecting 1 x 107 cells of a given
clone ~0.2 ml volume) into CAFl mice primed with
2,6,10,14-tetramethylpentadecane, sold by Aldrich Chemical
Company under the name Pristine. The malignant ascites
from these mice were then used to characterize lymphocytes
as described below in Example II. The subject hybrid
~5 antibody OKT5 was demonstrated by standard techniques to
be of IgGl subclass.
EXAMPLE II
Characterization of OKT5 Reactivity
A. Isolation of LvmphocYte Populations
Human peripheral blood mononuclear cells were isolated
from healthy volunteer donors (ages 15-40) by Ficoll-
Hypaque density gradient centrifugation (Pharmacia Fine
Chemicals, Piscataway, NJ) following the technique of
3Oyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97): 77,
1968. Unfractionated mononuclear cells were separated
* Trademark
~.~
,
ORTH3ss
117C~593
14
into surface Ig (B) and Ig (T plus Null) populations by
Sephadex G-200 anti-F(ab')2 column chromatography as
previously described by Chess, et al., J. Immunol.
113:1113 (1974). T cells were recovered by E rosetting
the Ig population with 5% sheep erythrocytes
(Microbiological Associates, Bethesda, MD). The rosetted
mixture was layered over Ficoll-Hypaque and the recovered
E pellet treated with 0O155M NH4Cl (10 ml per 108 cells).
The T cell population so obtained was ~2% EAC rosette
positive and ~95% E rosette positive as determined by
standard methods. In addition, the non-rosetting Ig
(Null cell) population was harvested from the Ficoll
interface, This latter population was ~5% E+and <2% sIg+.
The surface Ig+ (B) population was obtained from the
Sephadex G-200 column following elution with normal human
gamma globulin as previously described. This population
was >95% surface Ig+ and <5% E+.
Normal human macrophages were obtained from the mononu-
clear population by adherence to polystyrene. Thus, mono-
nuclear cells were resuspended in final culture media
(RPMI 1640~ 2.5mM HEPES [4-(2-hydroxyethyl)-1-piperazine-
propane sulfonic acid] buffer, 0.5% sodium bicarbonate,
200mM ~-glutamine, and 1% penicillin-streptomycin, supple-
mented with 20~ heat-inactivated human AB serum) at a
concentration of 2 x 106 cells and incubated in plastic
petri dishes (100 x 20 mm) (Falcon Tissue Culture Dish;
Falcon, Oxnard, CA) at 37C overnight. After extensive
washing to remove non-adherent cells, the adherent
population was detached by bris~ washing with cold
serum-free medium containing 2.5mM EDTA and occasional
scraping with the rubber tip of a disposable syringe
plunger. Greater than 85~ of the cell population w~s
capable of ingesting latex particles and had morphologic
characteristics of monocytes by Wright-Giemsa staining.
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~17(~593
B. Isolation of Thvmocytes
Normal human thymus gland was obtained from patients aged
two months to 14 years undergoing corrective cardiac
surgery. Freshly obtained portions of the thymus gland
were immediately placed in 5% fetal calf serum in medium
199 (Gibco), finely minced with forceps and scissors, and
subsequently made into single cell suspensions by being
pressed through wire mesh. The cells were next layered
over Ficoll-Hypaque and spun and washed as previously
described in section A above. The thymocytes so obtained
were ~95~ viable and ~90% E rosette positive.
C. Cell Line
An Epstein-Barr Virus (EBV) transformed B cell line from a
normal individual (Laz 156) was provided by Dr. H.
Lazarus, Sidney Farber Institute, Boston, MA.
EXAMPLE III
CYtofluorographic AnalYsis and Cell Separation
Cytofluorographic analysis of monoclonal antibodies with
all cell populations was performed by indirect immuno-
fluorescence with fluorescein-conjugated goat anti-mouse
IgG ~G/M FITC) (Meloy Laboratories) utilizing a Cytofluor-
ograf FC200/4800A ~Ortho Instruments). In brief, 1 x 10
cells were treated with 0.15 ml OKT5 at a 1:500 dilution,
incubated at 4C for 30 minutes, and washed twice. The
cells were then reacted with 0.15 ml of a 1:40 dilution
G/M FITC at 4C for 30 minutes, centrifuged, and washed
three times. Cells were then analyzed on the Cytofluoro-
graf, and the intensity of fluorescence per cell was
recorded on a pulse height analyzer. A similar pattern of
reactivity was seen at a dilution of 1:10,000, but further
dilution caused loss of reactivity. aackground staining
was obtained by substituting a 0.15 ml aliquot of 1:500
ascites from a CAFl mouse intraperitoneally injected with
a non-producing hybrid clone. Reactivity of lymphoid
populations with equine anti-TH2 and normal horse IgG were
~ ORTH 355
593
determined as previously described in the above-referenced
Reinherz, et. al., articles.
In experiments designed to separate T cell subsets, 100 x
106unfractionated T cells were labeled with 4 ml of a
1:500 dilution of OKT4 or OKTS and developed with G/M
FITC. OKT4 was previously shown to be reactive specifi-
cally with 55-60~ of peripheral blood T lymphocytes
representing the human helper subsets. By utilizing a
fluorescence activated cell sorter (F~CS-I) (Becton,
Dickinson, Mountain View, CA), T cells were separated into
OKT4 and OKT4 subsets as well as OKT5 and OKT5 subsets
from the same individual. Post sort viability was >95% by
Trypan blue exclusion in all instances. Purity of all
separated populations was ~95%.
EXAMPLE IV
Analysis of T cell subsets with Equine anti-TH2
In a similar fashion to Example III, equine anti-TH2 was
utilized to separate TH2+ and TH2 T cells on FACS by
labeling 60 x 10 unfractionated T cells with 0.12 ml
equine anti-TH2 and 0.1 ml of R/H FITC (Cappel
Laboratories, Downingtown, PA) as previously described by
Reinherz, et al. Purity and viabllity of sorted cells
were similar to the sorted populations described above.
FACS sorted subsets of T cells isolated with equine
anti-TH2, OKT4, or ORT5 were placed in culture for 48
hour~ with RPMI 1640 containing 20% human AB serum, 1%
penicillin-streptomycin, 200mM L-glutamine, 25mM HEPES
buffer (Microbiological Associates), and 0.5% sodium
bicarbonate at 37C in 5% C02 humid atmosphere. These
cultured cells were then analyzed on the Cytofluorograph
as described above. Background staining was determined by
substituting normal horse IgG for specific antibody and
staining as above.
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ll7ass3
EXAMPLE v
Functional Studies
A. Proliferative Studies
The mitogenic response of 105 unseparated and FACS-frac-
tionated T lymphocytes was tested in microculture to
optimal doses of Concanavalin A (Con A) (Calbiochem, La
Jolla, CA) and phyto hemagglutinin (PHA) (Burroughs-
Wellcome Company, Greenville, NC). Alloantigen prolifer-
ative response was measured concurrently for the same
populations with mitomycin treated Laz 156, an E~V
transformed human B lymphoblastoid cell line stimulus.
Proliferation to tetanus toxoid (Massachusetts Department
of Public Health Biological Laboratories, 80ston, MA) and
mumps antigen (Microbiological Associates) were tested
utilizing 10 ~g/ml final concentration and a 1:20
dilution, respectively. Five percent macrophages obtained
in the manner described above were added to all popula-
tions at the initiation of ln vitro cultures. Mitogen
stimulated cultures were pulsed after four days with 0.2
~Ci of 3H-thymidine ~3H-TdR; 1.9 Ci/mM specific activity)
(Schwarz/Mann, Division of Becton, Dickinson, Orangeburg,
NY) and harvested 18 hours later on a MASH II apparatus
~Microbiological Associates). H-TdR incorparation was
measured on a Packard Scintillation Counter (Pac~ard
Instrùment Company, Downer's Grove, IL). Background
H-TdR ~ncorporation was obtained by substituting media
for mitogen. Soluble and cell surface alloantigen
cultures were pulsed after five days with H-TdR for 18
hours, harvested, and counted as above.
B. Cvtotoxicitv Studies
Sensitization cultures for cell-mediated lympholysis (CML)
were established by placing unfractionated T cells with
mitomycin treated stimulator cells, all at 2 x 10
cells/ml in multiple microtiter plate wells. At the end
of five days, the unfractionated T cells were fractionated
into OKT5 and ORT5 T cell subsets on FACS. These T cell
_- subsets were then added to Cr sodium chromate labeled
target cells and specific cytotoxicity determined after a
~7~?S9 3 ORTH 355
18
six-hour cell incubation. Percent cytotoxicity was
determined with the following formula;
51Cr released by experiment - 51Cr released spontaneouslyx 100
51Cr released by freeze-thaw - 51Cr released spontaneously
All samples were performed in triplicate and results
expressed as the mean+ standard deviation.
C. Con A Activation of Suppressor Cells and Suppression
of MLC
-
Unfractionated T cells were activated with 20 ~g
Concanavalin A (Con A) (Calbiochem) per 10 cells. These
Con A treated cells were cultured upright in 25 cm surface
area tissue culture flasks (Falcon, Oxnard, CA) in RPMI
1640 (Grand Island Biological Company) containing 20%
human A8 serum, 1% penicillin-streptomycin, 200mM
L-glutamine, 25mM HEPES buffer (Microbiological Assoc-
iates), and 0.5% sodium bicarbonate for 48 hours at 37C
in a humid atmosphere containing 5~ CO2. Untreated
control T cells were cultured in an identical fashion but
without Con A. Subsequently, cells were spun, washed five
times, and added to fresh autologous responder cells in
one-way mixed lymphocyte culture (MLC) as described above.
~n suppression assay, one-way MLCs were established in
round-bottom microtiter plates (Linbro Chemical Company,
New Haven, CT) with triplicate wells, each containing 0.05
x 10 responder lymphocytes (whole mononuclear); 0.05 x l0
of either unactivated or Con A activated autologous
unractionated or fractionated T cells; and 0.1 x 10
mitomycin treated stimulated cells (Laz 156). After five
days, cultures were pulsed with 0.2 ~Ci of ~-TdR and
harvested 18 hours later, as above. Percent inhibition of
MLC proliferation was then calculated using the formula:
ORTH 355
~7~5~3
19
% inhibition = (l- cPmcpmn A ) ~ l00
where cpm Con A+ represents the results of 3H-TdR incor-
poration in MLC when Con A activated autologous T cells or
T cell subsets are added, and where cpm represents results
when unactivated autologous T cells are added.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l shows the fluorescence pattern obtained on the
Cytofluorograf after reacting normal human peripheral T
cells, B cells, Null cells, and macrophages with OKT5 at a
1:1000 dilution and G/M FITC in the upper row. For
comparison, results with equine anti-T~2 are shown in the
lower row.
Figure 2 shows the fluorescence pattern obtained on the
Cytofluorograf after reacting human thymocytes with OKT5
and G/M FITC(A) and with equine anti-T~2(B).
Figure 3 shows the fluorescence pattern obtained on the
Cytofluorograf after reacting equine anti-TH2 separated T
cell subsets with OKTS.
Figure 4 shows the fluorescence pattern obtained on the
Cytofluorograf after reacting OKT4 separated T cell
subsets with OKT5.
Figure 5 shows the cytotoxic capacity of unfractionated T
cells and T cell subsets separated with OKT5 following
allosensitization in MLC.
The production of the hybridoma and the production and
characterization of the resulting monoclonal antibody were
conducted as described in the above Examples. Although
large quantities of the subject antibody were prepared by
injecting the subject hybridoma intraperitoneally into
ORTH 355
7(r S93
mice and harvesting the malignant ascites, it is clearly
- contemplated that the hybridoma could be cultured in vitro
by techniques well-known in the art and the antibody
removed from the supernatant.
s
As shown in the upper row of Figure l, approximately 20~
of the human peripheral blood T cell population of a given
normal individual is reactive with OKT5, whereas the
entire B cell, NU11 cell, and macrophage populations
isolated from the same individual are unreactive with
OKTS. Similarly, equine anti-TH2reacts with 24~ of
peripheral T cells and is also unreactive with ~ cells,
Null cells and macrophages. The monoclonal antibody is
thus characterized in that it is reactive with an antigen
lS contained on the surface of approximately 20% of normal
human peripheral T cells, while being unreactive with any
antigens on the surface of the other three cell types
discussed above. As will be discussed below, the OKTS+
portion of the human peripheral T cell population is
~0 included in the cytotoxic and suppressor T cell subset.
This differential reactivity is one test by which the
subject antibody OKT5 may be detected and distinguished
from other antibodies.
As shown in Figure 2, approximately 80% of normal human
thymocytes from a six-month old infant are reactive with
ORT5. Similar results ~about 80% reactivity) were
obtained using additional thymus specimens from normal
individuals two months to l9 years of age. This value is
the same as for equine anti-TH2. The pattern of
reactivity in Figure 2 provides a second method of
detecting the subject antibody OKTS and distinguishing it
from other antibodies.
As shown in Figure 3, the subject antibody reacts with TR2+
but not with TH2 T cells. Approximately 5-10% of the TH2
subset was unreactive with ORT5. The pattern of reactiv-
ity in Figure 3 provides a third method of detecting the
~ ~7a'~ 5~ 3 ORT~ 355
subject antibody OKT5 and distinguishing it from other
antibodies.
As shown in Figure 4, the OKT4+ T cell subset is entirely
unreactive with OKTS. In contrast, the OKT4 T cell
subset is largely OKT5+ (6,800 of 10,000 cells tested).
These results indicate that the OKT5+ subset of T cells,
like the previously defined TH2+ subset, is reciprocal and
distinct from the OKT4+ subset. While the OKT4+ subset
contains the helper T cells, the OKTS+ subset (like the TH2+
subset) contains cytotoxic and suppressor T cells. This
pattern of reactivity in Figure 4 provides an additional
method of identifying OKTS antibody and distinguishing it
from other antibodies.
Figure 5 shows that the OKT5+ T cell subset effects CML.
The degree of lysis mediated by this population is greater
than that mediated by the unfractionated T cell popula-
tion. In contrast, the OKTS T cell population is mini-
mally lytic. Following activation in MLC against Laz 156,the unfractionated T cells were separated into OKTS+and
OKT5 subsets. Both unfractionated and fractionated T
cells were analyzed in CML against 5~r labeled Laz 156
targets at various effector:target ~E:T) ratios. The OKT5+
T cell subsets contained the effector population in
cell-mediated lympholysis. At E:T ratios of 5:1, 10:1,
and 20:1, the ORT5+ T cell population effected 40%, 58%,
and 77% specific lysis, respectively. The lytic capacity
of the isolated OKT5+ subset was considerably greater than
the unfractionated T cell population. Moreover, the ORT5-
T cell populatlon effected significantly less lysis at any
E :T ratio tested. Given the earlier observation that the
TH2 T cell subset in man effected CML, the present
findings would add support to the notion that the TH2+ and
OKT5 T cell subsets defined similar populations of
functionally active T lymphocytes. This differential
- ORTH 355
11.7(~5~3
cytotoxic capacity provides yet another method of
identifying ORT5 antibody and distinguishing it from other
antibodies.
Functional studies were conducted on lymphoid populations
which had been separated on a fluorescence activated cell
separator (FACS). The results of these studies are shown
in Tables I through III below and provide further support
for the previously described characterization of the
subject monoclonal antibody.
In these studies, an unfractionated T cell population was
treated with a 1:500 dilution of ORT5 and G/M FIT~ and
separated on the FACS in OKT5+ and OKT5 subsets. Given
the purity of the populations obtained (greater than or
equal to 95~), 5% macrophages were added to the separated
populations prior to in vitro culture. The unfractionated
T cell population and isolated OKT5+ and OKT5 T cell
subsets were then stimulated with PHA, Con A, soluble
antigens, and alloantigens to assess their in vitro
proliferative responses.
The proliferative response of the unfractionated T cell
populations to PHA and Con A is shown in Table I. A
maximal proliferative response by the unfractionated T
cell population is obtained with 1 ~g of PHA per 106 cells
with diminished responses occurring at 0.5 ~g and 0.1 ~g
of PHA per 106 cells. Treatment of the unfractionated T
cells with ORT5 and goat-mouse FITC without subsequent
fractionation did not alter the proliferative response.
In contra~t, differences in response to PHA were obtained
with the separated OKT5 and OKT5 T cell subset. The OKT5
population of cells responded to all doses of PHA in a
fashion similar to the unseparated T cell population.
However, the proliferative response of the OKT5 cells was
significantly less at all doses of PHA tested.
ORTH 355
` --` 117~593
Furthermore, at a dose of PHA of 0.1 ~g per 106 cells, the
ORT5+T cells did not proliferate at all, whereas t~e OKT5
T cell subset and unfractionated cells were still
responsive. The proliferative response of these subsets to
Con A, on the other hand, was similar and the two subsets
of cells could not be distinquished from one another or
the unfractionated T cell population.
The responses to alloantigen in MLC and to soluble anti-
gens were next examined. As shown in Table II, the
unfractionated T cell population, the unfractionatea T
cell population treated with OKT5 and G/M FI$C, and both
the ORT5+ and OKT5 T cell subsets responded in a similar
fashion in MLC against Laz 156. In contrast, proliferative
responses to soluble antigens provided the clearest
distinction between the subsets. In all cases tested, the
OKT5 T cell subset proliferated minimally to the soluble
antigens tetanus toxoid and mumps, whereas the OKT5 T
cell subset responded well.
Prior studies demonstrated that the TH2 population of T
lymphocytes could be induced with Con A to suppress
autologous lymphocytes in M~C. To determine whether OKT5
T cell subset could similarly express suppressor function,
T cells were activated or 48 hours with Con A and then
sorted with OKTS and OKT4 monoclonal antibodies into
distinct T cell subsets. Subsequently, these separated T
cell subpopulations were added to autologous responding
lymphocytes at the initiation of MLC. As shown in Table
III, unfractionated T cells activated with Con A suppres-
sed autologous cell prolieration in ~C. Treatment with
monoclonal antibody and G/M FI~C did not alter this
result. Although both OKT4 and OKT5 T cells prolifera-
ted to Con A, only OKT5 T cells became suppressive when
35 activated with Con A. The ORT4 subset was not suppres-
sive. In addition, the OKT5 subpopulation, which contains
both OKT4 and OKT4 TH2 subpopulations, was minimally
ORTH 355
;~17~5~3
24
suppressive ~hén compared to the highly suppressive OKT~
population.
Table IV shows the relationship between levels of periph-
eral T cells and T cell subsets and various diseasestates. These relationships may be used for diagnostic
purposes (e.g., to detect acute infectious mononucleosis)
by analyzing the blood sample of an individual suspected
of having one of these disease states to determine the
levels of T cells and T cell subsets. These relationships
may also be used for therapeutic purposes where the cause
of the disease state is an elevated level of a T cell
subset ~e.g., Type I acquired agammaglobulinemia). For
therapeutic use, administration of the appropriate
monoclonal antibody to a patient with an elevated T cell
subset level will decrease or eliminate the excess. The
relationships shown in Table IV are a further way in which
ORT5 antibody may be detected and distinguished from other
antibodies.
Other monoclonal antibody producing hybridomas prepared by
the present applicants (designated OKTl, ORT3, and QKT4)
are described and claimed in the following Cdn. patent
appllcations: SN 347,936, SN 34~,976 and SN 348,981
According to the present invention there are provided a
hybridoma capable of producing antibody against an antigen
found on human cytotoxic and suppressor T cells, a method
for producing this hybridoma, monoclonal antibody against
an antigen found on human cytotoxic and suppressor T
cells, methods for producing the antibody, and methods and
compositions for treatment or diagnosis of disease
employing this antibody.
B
- li7~93 ORTH 355
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- ORTH 355
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. ORTH 355
~.17~593
TABLE IV
PERIPHERAL T CELL LEVELS IN DISEASE STATES
~ Cell Levels
Disease State OKT3+ OKT4 ORT5
; Primary Biliary Cirrhosis (2) N +
Multiple Sclerosis (advanced - N
disease) (8)
Myasthemia Gravis ~early O O O
untreated) ~3)
Acute Graft vs Host (3)O to - - O
Acquired Agammaglobulinemia
Type I +
Type II
Hyper IgE (4) - N O to -
Acute Infectious Mono- + O to -- ++
nucleosis ~4)*
Hodgkins Disease
Stages I & II N N N
Stages III & IV -- N N
N ~ within normal limits
O - absent
+ - above normal
+~ = greatly above normal
- = below normal
-- = greatly below normal
* these levels return to normal about one week prior
to t~e disappearance of clinical symptoms
"The numbers in parentheses indicate the number of
patients evaluated."
-- ORTH 3ss
~:~7~S93
29
Although only a single hybridoma producing a single mono-
clonal antibody against human cytotoxic and suppressor T
cell antigen is described, it is contemplated that the
present inven~ion encompasses all monoclonal antibodies
exhibiting the characteristics described herein. It was
determined that the subject antibody OKT5 belongs to the
subclass IgGl, which is one of four subclasses of murine
IgG. These subclasses of immune globulin G differ from
one another in the so-called "fixed" regions, although an
antibody to a specific antigen will have a so-called
"variable~ region which is functionally identical regard-
less of which subclass of immune globulin G it belongs to.
That is, a monoclonal antibody exhibiting the charact-
eristic described herein may be of subclass IgGl, IgG2a,
lS IgG2b, or IgG3, or of classes IgM, IgA, or other known Ig
classes. The differences among these classes or sub-
classes will not affect the selectivity of the reaction
pattern of the antibody, but may affect the further
reaction of the antibody with other materials, such as
(for example) complement or anti-mouse antibodies.
Although the subject antibody i9 specifically IgGl, it is
contemplated that antibodies havLng the patterns of
reactivity illustrated herein are included within the
subject invention regardless of the immune globulin class
or subclass to which they belong.
Further included within the subject invention are methods
for preparing the monoclonal antibodies described above
employing the hybridoma technique illustrated herein.
Although only one example of a hybridoma is given herein,
it is contemplated that one skilled in the art could
follow the immunization, fusion, and selection methods
provided herein and obtain other hybridomas capable of
producing antibodies having the reactivity characteristics
described herein. Since the individual hybridoma produced
from a known mouse myeloma cell line and spleen cells from
- ORTH 355
- 1~7~593
a known species of mouse cannot be further identified
except by reference to the antibody produced by the
hybridoma, it is contemplated that all hybridomas pro-
ducing antibody having the reactivity characteristics
described above are included within the subject invention,
as are methods for making this antibody employing the
hybridoma.
Further aspects of the invention are methods of treatment
or diagnosis of disease employing the monoclonal antibody
OKT5 or any other monoclonal antibody exhibiting the
pattern of reactivity provided herein. The subject
antibody may be used to detect excess cytotoxic or sup-
pressor T cell activity by reacting a T cell composition
from an individual with OKT5 antibody. Excess cytotoxic
or suppressor T cell activity will be indicated by the
presence of more than 20-30~ of the total peripheral T
cell population which reacts with OKTS. This diagnostic
technique may be employed using OKT5 antibody alone or in
combination with other antibodies ~e.g., OKT3 and OKT4) as
shown in Table IV. Patterns of reactivity with a panel of
antibodies to T cells and T cell subsets will allow more
precise detection of certain disease states then is
possible using prior diagnostic methods.
Treatment of disease states (e.g., malignancies) mani-
festing themselves as an excess of suppressive activity
may be accomplished by administration of a therapeutically
effective amount of ORTS antibody to an individual in need
of such treatment. By selective reaction with suppressor
T cell antigen, the effective amount of OKT5 antibody will
reduce the excess of suppressor T cells, thus ameliorating
the effects of the suppressor T cell excess. Diagnostic
and therapeutic compositions comprising effective amounts
of OKTS antibody in admixture with diagnostically or
pharmaceutically acceptable carriers, respectively, are
also included within the present invention.
ORTH 355
1~7CS~3
The subject hybridoma OXT5 was deposited in the American
Type Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland 20852 on September 18, 1979, and September 28,
1979, and was given the ATCC accession number CRL 8013
and CRL 8016, respectively.
